Measles virus encoding a tumor antigen

ABSTRACT

The present invention relates to a recombinant virus of the family Paramyxoviridae comprising an expressible polynucleotide encoding at least one of (i) a tumor antigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii). The present invention further relates to a polynucleotide encoding said recombinant virus of the family Paramyxoviridae and to a host cell comprising said recombinant virus of the family Paramyxoviridae and/or said polynucleotide encoding said recombinant virus of the family Paramyxoviridae. Moreover, the present invention relates to a method for activating immune cells with antitumor activity in a sample comprising cancer cells and to further means, methods, and uses related to the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/784,895, filed Oct. 16, 2017, which claims priority to JP2016-203835, filed Oct. 17, 2016, which are hereby incorporated byreference it their entirety.

SEQUENCE LISTING

The Sequence Listing submitted herewith as an ASCII text file(2022-05-18_Continuation_Sequence_Listing.txt, created on May 16, 2022,78335 bytes) via EFS-Web is hereby incorporated by reference.

The present invention relates to a recombinant virus of the familyParamyxoviridae comprising an expressible polynucleotide encoding atleast one of (i) a tumor antigen, (ii) a fragment of a tumor antigen,and (iii) a variant of (i) or (ii). The present invention furtherrelates to a polynucleotide encoding said recombinant virus of thefamily Paramyxoviridae and to a host cell comprising said recombinantvirus of the family Paramyxoviridae and/or said polynucleotide encodingsaid recombinant virus of the family Paramyxoviridae. Moreover, thepresent invention relates to a method for activating immune cells withantitumor activity in a sample comprising cancer cells and to furthermeans, methods, and uses related to the present invention.

Oncolytic viruses (OV) which replicate selectively in tumor cells are anemerging modality of cancer treatment. Aside from direct cytopathiceffects and lysis of tumor cells, interactions of OV with the immunesystem can trigger systemic anti-tumor immunity. OV have been modifiedto express immunomodulatory transgenes to further enhance these effects(Melcher et al., Mol Ther. 2011, 19: 1008-1016). The vaccinia virusJX-594 and herpesvirus talimogene laherpavec (TVEC), both harboringGM-CSF, have shown promising results in clinical phase II and III trials(Heo et al., Nat Med. 2013,19: 329-336 and Andtbacka et al. J ClinOncol. 2013, 31, suppl; abstr LBA9008).

RNA viruses, in particular members of the family Paramyxoviridae like,e.g. measles virus, have also shown potential use in oncolysis. Virusesof the family Paramyxoviridae are negative-sense single-stranded RNAviruses and include human pathogens like, e.g. human parainfluenzaviruses, mumps virus, human respiratory syncytial virus, and measlesvirus. From wild type measles virus, several non-pathogenic strains,including a vaccine strain, have been derived, which have been shown toremain oncolytic. The measles virus vaccine strain has been developed asa vector platform to target multiple tumor entities and several clinicaltrials are ongoing (Russell et al., Nat Biotechnol. 2012, 30: 658-670).Recently, the capacity of oncolytic MV encoding GM-CSF to support theinduction of a specific anti-tumor immune response in terms of a tumorvaccination effect was demonstrated (Grossardt et al. Hum Gene Ther.2013, 24: 644-654.).

Tumor antigens, i.e. antigenic compounds associated with cancer cells,have been identified early in tumor research. Initially, the term “tumorantigen” was used to relate to antigens expressed by tumor cellsrelatively specifically, while the term “tumor specific antigen” wasused to relate to structures exclusively found on tumors. However, thisdistinction was later given up in view of the vast diversity ofexpression profiles in tumors. Tumor antigens have been known as tumormarkers, but also as targets useful for targeting cancer cells with highspecificity. MV oncolytic specificity can be achieved by entry-targetingbased on single-chain antibodies displayed on the viral attachmentprotein H blinded for its natural receptors CD46 and SLAM (Vongpunsawadet al. (2004), J Virol 78: 302; Nakamura et al. (2004), Nat Biotechnol22: 331). To date, a wide variety of tumor antigen-specific MV have beengenerated including vectors with specificity for CD20, CD38, CEA, PSCA,PSMA, EGFR, EGFRvIII, Her2neu, HMWAA (cf. Hammond et al. (2001), J Virol75(5):2087.(PMID:11160713); Peng et al. (2003), Blood 101(7):2557 (PMID:12433686); Allen et al. (2006), Cancer Res 66(24):11840 (PMID:17178881); Hasegawa et al. (2007), J Virol. 81(23):13149 (PMID:17804513); Ungerechts (2007), Cancer Res. 67(22):10939 (PMID: 18006839);Liu et al. (2009), Prostate 69(10):1128 (PMID: 19367568); Bossow et al.(2011), Cancer Gene Ther. 18(8):598 (PMID: 21701532); Zaoui et al.(2012), Cancer Gene Ther. 19(3):181-91 (PMID: 22076043); Kaufmann et al.(2013), J Invest Dermatol. 133(4):1034 (PMID: 23223133)).

There is, however, still a need in the art for improved cancertherapies, in particular for improved oncolytic viruses.

Accordingly, the present invention relates to a recombinant virus of thefamily Paramyxoviridae comprising an expressible polynucleotide encodingat least one of (i) a tumor antigen, (ii) a fragment of a tumor antigen,and (iii) a variant of (i) or (ii).

As used in the following, the terms “have”, “comprise” or “include” orany arbitrary grammatical variations thereof are used in a non-exclusiveway. Thus, these terms may both refer to a situation in which, besidesthe feature introduced by these terms, no further features are presentin the entity described in this context and to a situation in which oneor more further features are present. As an example, the expressions “Ahas B”, “A comprises B” and “A includes B” may both refer to a situationin which, besides B, no other element is present in A (i.e. a situationin which A solely and exclusively consists of B) and to a situation inwhich, besides B, one or more further elements are present in entity A,such as element C, elements C and D or even further elements.

Further, as used in the following, the terms “preferably”, “morepreferably”, “most preferably”, “particularly”, “more particularly”,“specifically”, “more specifically” or similar terms are used inconjunction with optional features, without restricting furtherpossibilities. Thus, features introduced by these terms are optionalfeatures and are not intended to restrict the scope of the claims in anyway. The invention may, as the skilled person will recognize, beperformed by using alternative features. Similarly, features introducedby “in an embodiment of the invention” or similar expressions areintended to be optional features, without any restriction regardingfurther embodiments of the invention, without any restrictions regardingthe scope of the invention and without any restriction regarding thepossibility of combining the features introduced in such way with otheroptional or non-optional features of the invention. Moreover, if nototherwise indicated, the term “about” relates to the indicated valuewith the commonly accepted technical precision in the relevant field,preferably relates to the indicated value±20%, more preferably ±10%,most preferably ±5%.

The term “recombinant virus”, as used herein, relates to a viruscomprising a genome modified by biotechnological means as compared toknown, naturally occurring, virus genomes. Preferably, the recombinantvirus is a virus comprising a genome modified as compared to naturallyoccurring virus genomes. Preferred biotechnological means for modifyinga viral genome are known to the skilled person and include any of themethods of molecular cloning, in particular recombinant DNA techniquesincluding, without limitation, cleavage of DNA by restriction enzymes,ligation of DNA, polymerase chain reaction (PCR), cloning of viralgenomes, and the like. It is understood by the skilled person thatviruses of the family Paramyxoviridae have a single-stranded (−)-RNA asa genome. Accordingly, the genome of the recombinant virus of thepresent invention, preferably, is obtained by cloning an expressionvector as described herein below comprising an expressible nucleotidesequence encoding said recombinant virus genome, followed by expressingsaid expressible nucleotide sequence encoding said recombinant virus ina permissive host cell. Alternatively, the recombinant virus genome mayalso be expressed in non-permissive host cells, e.g., preferably, fromrodents or other higher eukaryotes. Preferably, the recombinant virus ofthe present invention is a recombinant virus of the familyParamyxoviridae, more preferably a recombinant Morbillivirus, mostpreferably, a recombinant measles virus (MV). As will be understood bythe skilled person, the recombinant virus of the present invention maycomprise further modifications as compared to a naturally occurringvirus. Preferably, the recombinant virus comprises a polypeptidemediating a modified tropism and/or a polynucleotide encoding the same.More preferably, said polypeptide mediating a modified tropism is afusion polypeptide of a viral membrane integral polypeptide or of aviral membrane associated polypeptide with a polypeptide mediatingbinding to a target, e.g. a cell, preferably a specific kind of cell,more preferably a cancer cell. Preferably, said fusion polypeptidecomprises a viral hemagglutinin or a fragment thereof, preferably amembrane integral fragment thereof. Preferably, said fusion polypeptidecomprises a single-chain antibody specifically binding to a targetmolecule, e.g. to Carcinoembryonic antigen (CEA) or CD20. Mostpreferably, said fusion polypeptide is a fusion polypeptide of atruncated viral hemagglutinin with an anti-CD20 single-chain antibody orwith an anti-CEA single-chain antibody. Preferably, the recombinantvirus comprises a polynucleotide comprising the nucleic acid sequence ofany one of SEQ ID NOs: 3 to 5.

As used herein, the term “activator of the immune response” relates to acompound which, when contacted with a mixture of immune cells andimmune-response inducing cells, e.g. cancer cells, causes at least onetype of immune cell to be more active as compared to an immune cell ofthe same type comprised in the same mixture but lacking said compound.Preferably, the immune cell activated is a cell mediating a responseincreasing a subject's resistance to an antigen, i.e. preferably, saidimmune cell is not a tolerance-mediating immune cell. Preferably, theimmune cell activated by the activator of the immune response is aT-cell, more preferably a helper T-cell or a cytotoxic T-cell. Mostpreferably, the immune cell activated by the activator of the immuneresponse is a cytotoxic T-cell expressing PD-1. Measures of immune cellactivity are known to the skilled person and include, preferably,expression of activation markers, production of antibodies, excretion ofcytokines, and release of cytotoxins, e.g. perforin, granzymes, and/orgranolysin. As used herein, a tumor antigen does not have the activityof being an activator of the immune response; thus, preferably, a tumorantigen is not an activator of the immune response.

Preferably, the activator of the immune response is an activatorychemokine, preferably an activatory interleukin, more preferably IL-12.Also preferably, the activator of the immune response is an antagonistof a signaling pathway causing at least one type of immune cell tobecome inhibited. Accordingly, preferably, the activator of the immuneresponse is a ligand for an immune checkpoint blockade protein. Morepreferably, the activator of the immune response is a ligand for animmune checkpoint blockade protein. Still more preferably, the activatorof the immune response is an inhibitor of PD-1 receptor signaling. It isunderstood by the skilled person that signaling through a receptorsignaling pathway can be inhibited by either preventing the receptorfrom being activated, or by preventing the signal generated by theactivated receptor from being further transmitted. Accordingly,preferably, the activator of the immune response is a PD-L1 antagonist,the term “antagonist” relating to a compound binding to the molecule theeffect of which is antagonized and through said binding preventing saidmolecule from interacting with its native binding partner in aproductive, i.e. signaling-inducing, way. Preferred assays for saidactivity are described e.g. in WO 2015/128313 A1.

The term “immune response”, as used herein, relates to any protectiveresponse of the body of a subject to an antigen involving activity of atleast one type of leukocyte, preferably lymphocyte, and/or of at leastone antigen-recognizing macromolecule. Preferably, the immune responsecomprises inactivation of said antigen by production of antigen-specificantibodies (humoral immune response). More preferably, the immuneresponse comprises lysis of foreign cells or of body cells, preferablyof cancer cells, presenting said antigen (cell-mediated immuneresponse).

The term “modulating the immune response”, as used herein, relates toinducing a change in, preferably an activation of, the response of asubject's adaptive immune system. Accordingly, the term “modulating theimmune response”, preferably, relates to (i) newly inducing an immuneresponse of a subject to an antigen, (ii) to inducing an increase in thequality or intensity of the immune response of a subject to an antigen,and/or to (iii) redirecting an existing immune response to a target cellof interest, preferably a cancer cell, more preferably a cancer cellcomprising a tumor antigen. Preferably, modulating an immune responseincludes or is presenting an antigen or epitope on a cell in the contextof at least one of major histocompatibility complex (MHC) class I(MHC-I) and MHC class II (MHC-II) molecules. Preferably, the modulationis an activation, i.e. leads to an enhanced response to the antigen.Also preferably, modulating an immune response includes or is increasingthe frequency of antigen-specific T-cells in the blood of a subject.More preferably, modulating an immune response is increasing thefrequency of antigen-specific CD-4+ and/or CD-8+ T-cells in the blood ofsaid subject. Most preferably, modulating an immune response isincreasing the frequency of activated antigen-specific CD-4+ and/orCD-8+ T-cells in the blood of said subject. Preferably, said modulatingan immune response against an antigen induces killing of cellsexpressing said antigen by the immune system of the subject. Morepreferably, the term relates to inducing regression of at least onecancer comprising cells expressing said antigen. It is to be understoodthat inducing an immune response may not be effective in all subjects;e.g. in case a single specific epitope of a polypeptide is used as anantigen, a subject may lack MHC molecules suitable for effectivelydisplaying said epitope. The skilled person knows how to improve immunereaction in such case, e.g. by including further epitopes into therecombinant virus of the family Paramyxoviridae.

The term “antigen” is known to the skilled person and relates to achemical compound modulating an immune response in a host organism,wherein said host organism, preferably, is a vertebrate organism,preferably is a mammal, more preferably a human or an experimentalanimal, in particular a rat, a mouse, a rabbit, a guinea pig, a hamster,a sheep, a goat, a horse, a cow, a donkey, most preferably is a human.

Preferably, the antigen is an antigenic polypeptide. The term “antigenicpolypeptide”, as used herein, relates to a polypeptide comprising atleast one antigenic epitope. Preferably, said antigenic epitope is aglycan portion of a polypeptide. More preferably, said antigenic epitopeis a peptide and comprises a sequence of at least four, preferably atleast five, more preferably at least six, most preferably at least sevenamino acids. Also preferably, said antigenic epitope is a peptide andcomprises a sequence of from four to 15, preferably of from five totwelve, more preferably of from six to ten, most preferably of fromseven to nine amino acids. Preferably, the antigenic epitope is a T-cellepitope. A T-cell epitope, as is known to the one skilled in the art, isa contiguous sequence of amino acids comprised in a peptide, which canbe bound to a MHC class I or class II molecule to be presented on thesurface of a cell (MHC-I) or of a professional antigen presenting cell(MHC-II). The skilled artisan knows how to predict immunogenic peptidespresented on MHC-I or MHC-II (Nielsen et al., (2004), Bioinformatics, 20(9), 1388-1397), Bordner (2010), PLoS ONE 5(12): e14383) and how toevaluate binding of specific peptides (e.g. Bernardeau et al., (2011), JImmunol Methods, 371(1-2):97-105). Preferably, the antigenic epitope isan MHC-II epitope.

The term “cancer”, as used herein, relates to a disease of an animal,including man, characterized by uncontrolled growth by a group of bodycells (“cancer cells”). This uncontrolled growth may be accompanied byintrusion into and destruction of surrounding tissue and possibly spreadof cancer cells to other locations in the body. Preferably, alsoincluded by the term cancer is a relapse. Thus, preferably, the canceris a solid cancer, a metastasis, or a relapse thereof.

Preferably, the cancer is selected from the list consisting of acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, aids-related lymphoma, anal cancer, appendix cancer,astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer,bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma,carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma,chronic lymphocytic leukemia, chronic myelogenous leukemia, coloncancer, colorectal cancer, craniopharyngioma, endometrial cancer,ependymoblastoma, ependymoma, esophageal cancer, extracranial germ celltumor, extragonadal germ cell tumor, extrahepatic bile duct cancer,fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, gestational trophoblastic tumor, hairy cell leukemia,head and neck cancer, hepatocellular cancer, hodgkin lymphoma,hypopharyngeal cancer, hypothalamic and visual pathway glioma,intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma,medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouthcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer,oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarianepithelial cancer, ovarian germ cell tumor, ovarian low malignantpotential tumor, pancreatic cancer, papillomatosis, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sézary syndrome, small cell lung cancer, small intestine cancer,soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer,testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroidcancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer,waldenström macroglobulinemia, and wilms tumor. More preferably, thecancer is a solid cancer, a metastasis, or a relapse thereof. Mostpreferably, the cancer is a tumor derived from malignant melanoma, headand neck cancer, hepatocellular carcinoma, pancreatic carcinoma,prostate cancer, renal cell carcinoma, gastric carcinoma, colorectalcarcinoma, a lymphoma or a leukemia.

The term “tumor antigen”, as used herein, relates to any antigencomprised by a cancer cell. Thus, the term tumor antigen includesantigens comprised by cancer cells and by one or more non-cancer celltypes, e.g. CD19, CD20, CD22, CD30, and CD33. Preferably, the tumorantigen is a tumor-associated antigen, i.e. an antigen expressed bycancer cells, but not by normal cells of the same type as the cancercells in said subject. Also preferably, the tumor antigen is atumor-specific antigen, i.e. an antigen expressed by cancer cells, butnot by normal, i.e. non-cancer, cells of said subject at the givendevelopment stage. Also preferably, the tumor antigen is a neoantigen,i.e. an antigen not encoded in the germ line of said subject;preferably, said neoantigen is an antigen encoded by the tumor genome oran infectious agent, in particular a virus, preferably a tumorigenicvirus, more preferably a papillomavirus, an Epstein-Barr virus,Hepatitis B virus, or Hepatitis C virus; or said neoantigen is a muteincomprised by a cancer cell, i.e. a polypeptide variant produced after amutation of the encoding gene in the genome of said subject hasoccurred. As specified herein above, an antigen, and also a tumorantigen, has the biological activity of modulating an immune response ina host organism. Preferably, the tumor antigen has the biologicalactivity of modulating an immune response in a human. Also preferably,the tumor antigen has the biological activity of modulating an immuneresponse in the subject from which the cells comprising said tumorantigen are derived from. Preferably, the tumor antigen is a biologicalmacromolecule, more preferably a polypeptide. Preferably, the tumorantigen is a human tumor antigen.

Preferably, the tumor antigen is selected from the group consisting ofL-dopachrome-tautomerase (TRP2), melanocyte protein PMEL (gp100), HPVE6/7, MAGE 1, MAGE 3, NY-ESO, androgen receptor (AR), BCL-1,calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial celladhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogenreceptors (mER), FAP HER2/neu, human high molecular weightmelanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21, MOC-52,melan-A/MART-1, melanoma-associated antigen, mucin, OKT9, progesteronereceptor (PGR), prostate specific antigen (PSA), prostate stem cellantigen (PSCA), prostate-specific membrane antigen (PSMA),symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33. More preferably,the tumor antigen is HPV E6/7, MAGE 1, MAGE 3, NY-ESO, TRP2 or gp100,more preferably is TRP2. The open reading frame encoding human TRP2 isdisclosed herein as SEQ ID NO: 1, the amino acid sequence of human TRP2is disclosed as SEQ ID NO:2.

Preferably, the recombinant virus of the family Paramyxoviridaecomprises an expressible polynucleotide encoding a fragment of a tumorantigen. As used herein, the term “fragment of a tumor antigen” relatesto a substructure of a tumor antigen having the biological activity ofmodulating an immune response as specified herein above. Thus, thefragment of a tumor antigen, preferably, is an antigenic polypeptide asspecified herein, comprising at least a subsequence of said tumorantigen polypeptide; preferably, the fragment of a tumor antigencomprises at least one antigenic epitope in such case, more preferablyat least one epitope which is antigenic in the subject the cellscomprising the tumor antigen are derived from. Preferably, a fragment ofa tumor antigen is or is derived from, e.g., a degradation product or asplice variant of the tumor antigen. Preferably, the fragment of a tumorantigen comprises, preferably consists of: (i) a fragment of a humanpapillomavirus (HPV) E6 polypeptide, preferably of an E6 polypeptide ofa high-risk HPV, e.g. of a HPV16 E6 (Genbank Acc No: NP_041325.1GI:9627104), (ii) a fragment of a HPV E7 polypeptide, preferably of anE7 polypeptide of a high-risk HPV, e.g. of a HPV16 E7 (Genbank Acc No:NP_041326.1 GI:9627105); (iii) TRP2, preferably human TRP2 (preferably.encoded by Genbank Acc No: NM 001922.4 GI:1015809739), (iv)cancer/testis antigen 1B (CTAG1B, also referred to as NY-ESO, preferablyencoded by Genbank Acc No: NM 001327.2 GI:215272337, or (v) an arbitrarycombination of any of (i) to (iv).

Preferably, the recombinant virus of the family Paramyxoviridaecomprises an expressible polynucleotide encoding a variant of a tumorantigen and/or of a fragment of a tumor antigen. As used herein, theterm “variant” of a tumor antigen relates to an antigen beingnon-identical to said tumor antigen having the activity of modulatingthe immune response. Thus, as used herein, the term polypeptide“variant” relates to any chemical molecule comprising at least onepolypeptide or fusion polypeptide as specified elsewhere herein, havingthe indicated activity, but differing in primary structure from saidpolypeptide or fusion polypeptide. Thus, the polypeptide variant,preferably, is a mutein having the indicated activity. Preferably, thepolypeptide variant comprises a peptide having an amino acid sequencecorresponding to an amino acid sequence of 5 to 200, more preferably 6to 100, even more preferably 7 to 50, or, most preferably, 8 to 30consecutive amino acids comprised in a polypeptide as specified above.Moreover, also encompassed are further polypeptide variants of theaforementioned polypeptides. Such polypeptide variants have at leastessentially the same biological activity as the specific polypeptides.Moreover, it is to be understood that a polypeptide variant as referredto in accordance with the present invention shall have an amino acidsequence which differs due to at least one amino acid substitution,deletion and/or addition, wherein the amino acid sequence of the variantis still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% identical with the amino acid sequence of the specificpolypeptide. The degree of identity between two amino acid sequences canbe determined by algorithms well known in the art. Preferably, thedegree of identity is to be determined by comparing two optimallyaligned sequences over a comparison window, where the fragment of aminoacid sequence in the comparison window may comprise additions ordeletions (e.g., gaps or overhangs) as compared to the sequence it iscompared to for optimal alignment. The percentage is calculated bydetermining, preferably over the whole length of the polypeptide, thenumber of positions at which the identical amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Optimal alignment of sequences forcomparison may be conducted by the local homology algorithm of Smith andWaterman (1981), by the homology alignment algorithm of Needleman andWunsch (1970), by the search for similarity method of Pearson and Lipman(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by visual inspection. Given that two sequences have been identifiedfor comparison, GAP and BESTFIT are preferably employed to determinetheir optimal alignment and, thus, the degree of identity. Preferably,the default values of 5.00 for gap weight and 0.30 for gap weight lengthare used. Polypeptide variants referred to herein may be allelicvariants or any other species specific homologs, paralogs, or orthologs;moreover, the polypeptide variants referred to herein include fragmentsof the specific polypeptides or the aforementioned types of polypeptidevariants as long as these fragments and/or variants have the biologicalactivity as referred to above. Further included are variants whichdiffer due to posttranslational modifications such as phosphorylation,glycosylation, ubiquitinylation, sumoylation, or myristylation, byincluding non-natural amino acids, and/or by being peptidomimetics.Preferably, the variant of the tumor antigen or fragment of a tumorantigen comprises, preferably consists of, at least one of a variant of(i) a HPV E6 polypeptide, preferably of an E6 polypeptide of a high-riskHPV, e.g. of a HPV16 E6 (Genbank Acc No: NP_041325.1 GI:9627104), (ii) aHPV E7 polypeptide, preferably of an E7 polypeptide of a high-risk HPV,e.g. of a HPV16 E7 (Genbank Acc No: NP_041326.1 GI:9627105); (iii) TRP2,preferably human TRP2 (preferably. encoded by Genbank Acc No: NM001922.4 GI:1015809739), (iv) cancer/testis antigen 1B (CTAG1B, alsoreferred to as NY-ESO, preferably encoded by Genbank Acc No: NM 001327.2GI:215272337), or (v) an arbitrary combination of any of (i) to (iv).

Preferably, the tumor antigen, fragment of a tumor antigen, or variantof tumor antigen is a fusion polypeptide comprising further amino acidsor polypeptides. More preferably, the fusion polypeptide furthercomprises a detectable tag. The term “detectable tag” refers to astretch of amino acids which are added to or introduced into the fusionpolypeptide. Preferably, the tag is added C— or N— terminally to thefusion polypeptide. The said stretch of amino acids shall allow fordetection of the fusion polypeptide by an antibody which specificallyrecognizes the tag or it shall allow for forming a functionalconformation, such as a chelator or it shall allow for visualization byfluorescent tags. Preferred tags are the Myc-tag, FLAG-tag, 6-His-tag,HA-tag, GST-tag or GFP-tag. These tags are all well known in the art. Ina further preferred embodiment, said fusion protein comprises a peptideor polypeptide comprising the amino acid sequence of a further tumorantigen or of an activator of the immune response.

The term “expressible polynucleotide”, as used herein, relates to apolynucleotide operatively linked to at least one expression controlsequence causing transcription of the nucleic acid sequence comprised insaid polynucleotide to occur, preferably in eukaryotic cells or isolatedfractions thereof, preferably into a translatable mRNA or into a viralgenome. Regulatory elements ensuring expression in eukaryotic cells,preferably mammalian cells, are well known in the art. They, preferably,comprise regulatory sequences ensuring initiation of transcription and,optionally, poly-A signals ensuring termination of transcription andstabilization of the transcript. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Preferably,the aforesaid at least one expression control sequence is an expressioncontrol sequence of a (−)strand RNA virus, more preferably of aParamyxovirus as described herein above, most preferably of an MV. Thus,preferably, at least one expression control sequence comprises a(−)strand RNA viral regulatory sequence ensuring initiation oftranscription (consensus “gene start signal”, preferably consensus MV“gene start signal”) and termination signals (consensus “gene stopsignal”, preferably, consensus MV “gene stop signal”) ensuringtermination of transcription and stabilization of the transcript. It isknown in the art that production of viral particles in permissive hostcells can be initiated by transfecting into said permissive host cellsone or more expressible DNA constructs encoding (i) a recombinant viralanti-genome, (ii) the viral L gene, (iii) the viral P gene, and (iv) theviral N gene. It is also understood by the skilled person that, once aviral genome and the aforesaid viral genes were expressed in said hostcell, replication and assembly of viral particles occurs in thecytoplasm of the host cell and is, therefore, solely dependent on viralregulatory signals. Preferably, production of viral particles inpermissive host cells may also be initiated by transfecting into saidpermissive host cells one or more expressible DNA (i) a constructencoding a recombinant viral anti-genome and introducing into saidpermissive host cells the polypeptide products of (ii) the viral L gene,(iii) the viral P gene, and (iv) the viral N gene. The termpolynucleotide, as used herein, preferably encompasses polynucleotidevariants as specified elsewhere herein. Preferably, the expressiblepolynucleotide encoding the polynucleotides of the invention, preferably(i) a tumor antigen, (ii) a fragment of a tumor antigen, or (iii) avariant of (i) or (ii), is comprised in the genome of the recombinantvirus of the family Paramyxoviridae in a region corresponding to theregion intervening the H and the L gene of measles virus. Preferably,the expressible polynucleotide comprises further coding sequences. Morepreferably, the expressible polynucleotide encodes an activator of theimmune response. In an embodiment, the expressible polynucleotideencodes at least one further of (i) a tumor antigen, (ii) a fragment ofa tumor antigen, and (iii) a variant of (i) or (ii). As will beunderstood, the further polypeptides encoded may be encoded as separatepolypeptides or as fusion polypeptides comprising at least one,preferably at least two, more preferably at least three of (i) a tumorantigen, (ii) a fragment of a tumor antigen, and (iii) a variant of (i)or (ii). Thus, preferably, the expressible polynucleotide may encode amultitude of antigenic epitopes of tumor antigens as one or more fusionpolypeptide(s). In a further embodiment, said activator of the immuneresponse comprises, preferably is, a secreted soluble activator of theimmune response, preferably an immunoglobulin or fragment thereof, morepreferably a secreted immunoglobulin or fragment thereof.

As used herein, the term “immunoglobulin” relates to a polypeptide beinga soluble immunoglobulin, preferably an antibody from any of the classesIgA, IgD, IgE, IgG, or IgM, preferably having the activity of binding,more preferably specifically binding, a molecule of interest.Immunoglobulins against antigens of interest can be prepared bywell-known methods using, e.g., a purified molecule of interest or asuitable fragment derived therefrom as an antigen. A fragment which issuitable as an antigen may be identified by antigenicity determiningalgorithms well known in the art. Such fragments may be obtained eitherfrom one of the molecules of interest by proteolytic digestion, may be asynthetic peptide, or may be obtained by recombinant expression.Preferably, a peptide of a molecule of interest used as an antigen islocated at the exterior of a cell expressing the molecule of interest;i.e. preferably, the epitope the binding domain interacts with,preferably, is an extracellular domain. Preferably, the immunoglobulinof the present invention is a monoclonal antibody, a human or humanizedantibody or primatized, chimerized antibody or a fragment thereof, solong as they exhibit the desired binding activity as specified elsewhereherein. Also comprised as antibodies of the present invention are abispecific antibody, a synthetic antibody, or a chemically modifiedderivative of any of these. Preferably, the antibody of the presentinvention shall specifically bind (i.e. does only to a negligible extentor, preferably, not cross react with other polypeptides or peptides) toa molecule of interest as specified above. Specific binding can betested by various well known techniques. Antibodies or fragments thereofcan be obtained by using methods which are described, e.g., in Harlowand Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold SpringHarbor, 1988. Monoclonal antibodies can be prepared by the techniquesoriginally described in Kohler and Milstein, Nature. 1975. 256: 495; andGalfré, Meth. Enzymol. 1981, 73: 3, which comprise the fusion of mousemyeloma cells to spleen cells derived from immunized mammals. As will beunderstood by the skilled person, a molecule of interest, bound by animmunoglobulin of the present invention, may also be an Fc receptor or acomplement protein binding an Fc part of an antibody; accordingly, theimmunoglobulin preferably is an Fc domain of an antibody, morepreferably a soluble Fc domain of an antibody, most preferably asecreted soluble Fc domain of an antibody. Preferably, said antibody theFc domain is derived from is an IgG, more preferably an IgG1, mostpreferably a human IgG1.

“Immunoglobulin fragments” comprise a portion of an intactimmunoglobulin, preferably of an antibody, in an embodiment, comprisethe antigen-binding region thereof. Examples of antibody fragments andfusion proteins of variable regions include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; single-domain-antibodies (VHH), also known as nanobodies, andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen. “Fv” is the minimum antibody fragment which contains a completeantigen-binding site. Preferably, a two-chain Fv species consists of adimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three hypervariableregions (HVRs, also referred to as complementarity determining regions(CDRs)) of each variable domain interact to define an antigen-bindingsite. Collectively, the six HVRs of one scFv confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three HVRs specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site. The term “diabodies” refers to antibodyfragments with two antigen-binding sites, which fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) in the same polypeptide chain (VH-VL). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies may bebivalent or bispecific. Diabodies are described more fully in, forexample, EP 0 404 097; WO 1993/01161; Hudson et al., Nat. Med. 9 (2003)129-134; and Hollinger et al., PNAS USA 90 (1993) 6444-6448. Triabodiesand tetrabodies are also described in Hudson et al., Nat. Med. 9 (2003)129-134.

The term “secreted”, as used herein, relates to a compound beingtransferred from the interior of a host cell to the exterior of saidhost cell by a mechanism intrinsic to said host cell. Preferably,secretion of a polypeptide or fusion polypeptide is mediated by a,preferably eukaryotic, signal peptide mediating import of said peptideor polypeptide into the lumen of the endoplasmic reticulum and, morepreferably, by the absence of retention signals. Signal peptides causingsecretion of peptides or polypeptides are known in the art. Preferably,the signal peptide is an IL-12 signal peptide. Also preferably, thesignal peptide is or comprises an Ig leader sequence. More preferably,the signal peptide is or comprises a human Ig leader sequence. Stillmore preferably, the signal peptide is or comprises a matching leadersequence, i.e. a leader sequence selected from the same Ig kappasubgroup as the variable light chain of the antibody, preferably, of thesingle-chain antibody.

Advantageously, it was found in the work underlying the presentinvention that recognition of tumor antigens not recognized by theimmune system can be induced by presenting said tumor antigens in thecontext of a virus infection. Without wishing to be bound by theory, itis presumed that expression of tumor antigens in the context of ameasles virus infection breaks tumor-induced tolerance and makes andimmune response to the tumor antigen possible. Moreover, it was foundthat the expression of tumor antigens on tumor cells induced by measlesvirus infection increases infiltration and persistence of T-cells intumors.

The definitions made above apply mutatis mutandis to the following.Additional definitions and explanations made further below also applyfor all embodiments described in this specification mutatis mutandis.

The present invention further relates to polynucleotide encoding therecombinant virus of the family Paramyxoviridae according to the presentinvention

The term “polynucleotide” is understood by the skilled person to relateto a polymer composed of a series of contiguous nucleotides; the termencompasses single as well as double stranded polynucleotides.Preferably, comprised are also chemically modified polynucleotidesincluding naturally occurring modified polynucleotides such asglycosylated or methylated polynucleotides or artificial modified onessuch as biotinylated polynucleotides and polynucleotides comprisingmodified nucleotides. Preferably, the nucleotides comprised in thepolynucleotide are naturally occurring nucleotides. Preferably, thepolynucleotide is RNA, including mRNA, or DNA, including cDNA. Morepreferably, the polynucleotide is DNA. Preferably, the nucleotidescomprised in the polynucleotide comprise, preferably compriseexclusively, the bases adenine, guanine, cytosine, and thymine in casethe polynucleotide is DNA; also preferably, the nucleotides comprised inthe polynucleotide comprise, preferably comprise exclusively, the basesadenine, guanine, cytosine, and uracil in case the polynucleotide isRNA. The polynucleotides of the present invention either consist of,essentially consist of, or comprise the indicated nucleic acidsequences. Thus, they may contain further nucleic acid sequences aswell. The polynucleotide of the present invention shall be provided,preferably, either as an isolated polynucleotide (i.e. isolated from itsnatural context) or in genetically modified form.

The term “polynucleotide encoding a recombinant virus”, as used herein,relates to a polynucleotide comprising a nucleic acid sequence ornucleic acid sequences sufficient for generating a virus particle or avirus-like particle in a host cell. It is understood by the skilledperson that a virus is constituted by a polynucleotide genome and atleast one kind of capsid polypeptide. Accordingly, the polynucleotideencoding a recombinant virus of the present invention, preferably,comprises a recombinant virus genome. As will be understood by theskilled person, in case the polynucleotide encoding a recombinant virusis comprised in a virus according to the present invention, i.e. a virusof the family Paramyxoviridae, the polynucleotide is (−)strand RNA. Itis also understood by the skilled person that in case the polynucleotideis DNA comprised in a host cell, at least an RNA-dependent RNApolymerase activity will additionally be required to produce viralparticles from said DNA polynucleotide. Preferably, the polynucleotideencoding a recombinant virus comprises or consists of the nucleic acidsequence as specified elsewhere herein. As annotated herein, thesequence of the DNA copy of negative-strand (−)RNA viruses is annotatedin the usual 5′→>3′-orientation; this corresponds to the viral sequencein antigenomic (+)RNA orientation with respect to the natural3′→5′-orientation of negative-strand (−)RNA viruses. Preferably, thepolynucleotide encoding a recombinant virus is based on Measles virusstrain Edmonston (Moraten vaccine), Genbank Acc No: AF266287.1GI:9181873. More preferably, the polynucleotide encoding a recombinantvirus comprises, preferably consists of, the nucleic acid sequence ofany one of SEQ ID NOs: 3 to 5.

The term polynucleotide, preferably, includes polynucleotide variants.The term “polynucleotide variant”, as used herein, relates to a variantof a polynucleotide related to herein comprising a nucleic acid sequencecharacterized in that the sequence can be derived from theaforementioned specific nucleic acid sequence by at least one nucleotidesubstitution, addition and/or deletion, wherein the polynucleotidevariant shall have the activity as specified for the specificpolynucleotide. Preferably, said polynucleotide variant is an ortholog,a paralog or another homolog of the specific polynucleotide. Alsopreferably, said polynucleotide variant is a naturally occurring alleleof the specific polynucleotide. Polynucleotide variants also encompasspolynucleotides comprising a nucleic acid sequence which is capable ofhybridizing to the aforementioned specific polynucleotides, preferably,under stringent hybridization conditions. These stringent conditions areknown to the skilled worker and can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. Apreferred example for stringent hybridization conditions arehybridization conditions in 6×sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C. The skilled worker knows that thesehybridization conditions differ depending on the type of nucleic acidand, for example when organic solvents are present, with regard to thetemperature and concentration of the buffer. For example, under“standard hybridization conditions” the temperature differs depending onthe type of nucleic acid between 42° C. and 58° C. in aqueous bufferwith a concentration of 0.1× to 5×SSC (pH 7.2). If organic solvent ispresent in the abovementioned buffer, for example 50% formamide, thetemperature under standard conditions is approximately 42° C. Thehybridization conditions for DNA:DNA hybrids are preferably for example0.1×SSC and 20° C. to 45° C., preferably between 30° C. and 45° C. Thehybridization conditions for DNA:RNA hybrids are preferably, forexample, 0.1×SSC and 30° C. to 55° C., preferably between 45° C. and 55°C. The abovementioned hybridization temperatures are determined forexample for a nucleic acid with approximately 100 bp (=base pairs) inlength and a G+C content of 50% in the absence of formamide. The skilledworker knows how to determine the hybridization conditions required byreferring to textbooks such as the textbook mentioned above, or thefollowing textbooks: Sambrook et al., “Molecular Cloning”, Cold SpringHarbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press, Oxford.Alternatively, polynucleotide variants are obtainable by PCR-basedtechniques such as mixed oligonucleotide primer-based amplification ofDNA, i.e. using degenerated primers against conserved domains of apolypeptide of the present invention. Conserved domains of a polypeptidemay be identified by a sequence comparison of the nucleic acid sequenceof the polynucleotide or the amino acid sequence of the polypeptide ofthe present invention with sequences of other organisms. As a template,DNA or cDNA from bacteria, fungi, or plants preferably, from animals maybe used. Further, variants include polynucleotides comprising nucleicacid sequences which are at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%identical to the specifically indicated nucleic acid sequences.Moreover, also encompassed are polynucleotides which comprise nucleicacid sequences encoding amino acid sequences which are at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequencesspecifically indicated. The percent identity values are, preferably,calculated over the entire amino acid or nucleic acid sequence region. Aseries of programs based on a variety of algorithms is available to theskilled worker for comparing different sequences. In this context, thealgorithms of Needleman and Wunsch or Smith and Waterman giveparticularly reliable results. To carry out the sequence alignments, theprogram PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al.,CABIOS, 5 1989: 151-153) or the programs Gap and BestFit (Needleman andWunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv.Appl. Math. 2; 482-489 (1981))), which are part of the GCG softwarepacket (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711 (1991)), are to be used. The sequence identity values recitedabove in percent (%) are to be determined, preferably, using the programGAP over the entire sequence region with the following settings: GapWeight: 50, Length Weight: 3, Average Match: 10.000 and AverageMismatch: 0.000, which, unless otherwise specified, shall always be usedas standard settings for sequence alignments.

A polynucleotide comprising a fragment of any of the specificallyindicated nucleic acid sequences is also encompassed as a variantpolynucleotide of the present invention. The fragment shall still encodea polypeptide or fusion polypeptide which still has the activity asspecified. Accordingly, the polypeptide encoded may comprise or consistof the domains of the polypeptide of the present invention conferringthe said biological activity. A fragment as meant herein, preferably,comprises at least 50, at least 100, at least 250 or at least 500consecutive nucleotides of any one of the specific nucleic acidsequences or encodes an amino acid sequence comprising at least 20, atleast 30, at least 50, at least 80, at least 100 or at least 150consecutive amino acids of any one of the specific amino acid sequences.

The present invention further relates to a host cell comprising therecombinant virus of the family Paramyxoviridae according to the presentinvention and/or the polynucleotide encoding the recombinant virus ofthe family Paramyxoviridae according to the present invention.

As used herein, the term “host cell” relates to a vertebrate cell.Preferably, the cell is a vertebrate cell, preferably is a mammaliancell, more preferably a human cell or a cell of an experimental animal,in particular a rat, a mouse, a rabbit, a guinea pig, a hamster, asheep, a goat, a horse, a cow, a donkey cell, most preferably is a humancell. Preferably, the host cell is a cancer cell, more preferably atumor cell.

Further, the present invention relates to a medicament comprising

(a) (i) a recombinant virus of the family Paramyxoviridae according tothe present invention,

-   -   (ii) a polynucleotide encoding the recombinant virus of the        family Paramyxoviridae according to (i), preferably a        polynucleotide according to an embodiment as specified herein        below;    -   (iii) a host cell comprising the recombinant virus of the family        Paramyxoviridae according to (i) and/or a polynucleotide        encoding the recombinant virus of the family Paramyxoviridae        according to (ii); preferably a host cell according to an        embodiment as specified herein below; or    -   (iv) any combination of (i) to (iii); and

(b) at least one pharmacologically acceptable excipient.

The terms “medicament” and “pharmaceutical composition”, as used herein,relate to the compounds of the present invention and optionally one ormore pharmaceutically acceptable carrier, i.e. excipient. The compoundsof the present invention can be formulated as pharmaceuticallyacceptable salts. Acceptable salts comprise acetate, methyl ester, HCl,sulfate, chloride and the like. The pharmaceutical compositions are,preferably, administered locally, topically or systemically. Suitableroutes of administration conventionally used for drug administration areoral, intravenous, or parenteral administration as well as inhalation. Apreferred route of administration is intra-tumoral administration.However, depending on the nature and mode of action of a compound, thepharmaceutical compositions may be administered by other routes as well.For example, polynucleotide compounds may be administered in a genetherapy approach by using viral vectors or viruses or liposomes.

Moreover, the compounds can be administered in combination with otherdrugs either in a common pharmaceutical composition or as separatedpharmaceutical compositions wherein said separated pharmaceuticalcompositions may be provided in form of a kit of parts. The compoundsare, preferably, administered in conventional dosage forms prepared bycombining the drugs with standard pharmaceutical carriers according toconventional procedures. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation. It will be appreciated that the form andcharacter of the pharmaceutically acceptable carrier or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The excipient(s) must be acceptable in the sense of being compatiblewith the other ingredients of the formulation and being not deleteriousto the recipient thereof. The excipient employed may be, for example, asolid, a gel or a liquid carrier. Exemplary of solid carriers arelactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary of liquidcarriers are phosphate buffered saline solution, syrup, oil such aspeanut oil and olive oil, water, emulsions, various types of wettingagents, sterile solutions and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. The diluent(s) is/are selected so as notto affect the biological activity of the combination. Examples of suchdiluents are distilled water, physiological saline, Ringer's solutions,dextrose solution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, non-immunogenic stabilizers and the like.

A therapeutically effective dose refers to an amount of the compounds tobe used in a pharmaceutical composition of the present invention whichprevents, ameliorates or treats the symptoms accompanying a disease orcondition referred to in this specification. Therapeutic efficacy andtoxicity of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician andother clinical factors; preferably in accordance with any one of theabove described methods. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment. A typical dose can be, for example, in the rangeof 1 to 1000 μg for a polypeptide or polynucleotide, or 104-108 viralparticles for a virus or a virus-like particle; however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. Progress can be monitored by periodicassessment. The pharmaceutical compositions and formulations referred toherein are administered at least once in order to treat or ameliorate orprevent a disease or condition recited in this specification. However,the said pharmaceutical compositions may be administered more than onetime, for example from one to four times daily up to a non-limitednumber of days. Specific pharmaceutical compositions are prepared in amanner well known in the pharmaceutical art and comprise at least oneactive compound referred to herein above in admixture or otherwiseassociated with a pharmaceutically acceptable carrier or diluent. Formaking those specific pharmaceutical compositions, the activecompound(s) will usually be mixed with a carrier or the diluent, orenclosed or encapsulated in a capsule, sachet, cachet, paper or othersuitable containers or vehicles. The resulting formulations are to beadapted to the mode of administration, i.e. in the forms of tablets,capsules, suppositories, solutions, suspensions or the like. Dosagerecommendations shall be indicated in the prescribers or usersinstructions in order to anticipate dose adjustments depending on theconsidered recipient.

The present invention further relates to a method for activating immunecells with antitumor

-   -   activity in a sample comprising cancer cells and immune cells,        comprising    -   a) contacting said sample comprising cancer cells and immune        cells with        -   (i) a recombinant virus of the family Paramyxoviridae            according to the present invention,        -   (ii) a polynucleotide encoding the recombinant virus of the            family Paramyxoviridae according to (i), preferably a            polynucleotide according to an embodiment as specified            herein below;        -   (iii) a host cell comprising the recombinant virus of the            family Paramyxoviridae according to (i) and/or a            polynucleotide encoding the recombinant virus of the family            Paramyxoviridae according to (ii); preferably a host cell            according to an embodiment as specified herein below; or        -   (iv) any combination of (i) to (iii); and thereby,    -   b) activating immune cells with antitumor activity comprised in        said sample.

The method for activating immune cells of the present invention,preferably, may comprise steps in addition to those explicitly mentionedabove. For example, further steps may relate, e.g., to providing asample for step a), or administration of additional compounds to theimmune cells, e.g. immunostimulatory compounds before or during step b).Moreover, one or more of said steps may be performed by automatedequipment. The method for activating immune cells of the presentinvention, preferably, is an in vitro method.

The term “contacting”, as used in the context of the methods of thepresent invention, is understood by the skilled person. Preferably, theterm relates to bringing a compound, e.g. a virus, a sample, or asubject of the present invention in physical contact with a furthercompound and thereby allowing the compound and the further compound tointeract.

The term “immune cells”, as used herein, relates cells mediating animmune response in a subject. Preferably, the immune cell is aleukocyte, preferably a lymphocyte. Preferably, the immune cell is aB-cell; more preferably, the immune cell is a T-cell, still morepreferably a cytotoxic T cell or a T helper cell.

The present invention further relates to a method for treating cancer ina subject afflicted with cancer, comprising

-   -   1. a) contacting said subject with        -   2. (i) a recombinant virus of the family Paramyxoviridae            according to the present invention,        -   3. (ii) a polynucleotide encoding the recombinant virus of            the family Paramyxoviridae according to (i), preferably a            polynucleotide according to an embodiment as specified            herein;        -   4. (iii) a host cell comprising the recombinant virus of the            family Paramyxoviridae according to (i) and/or a            polynucleotide encoding the recombinant virus of the family            Paramyxoviridae according to (ii); preferably a host cell            according to an embodiment as specified herein; or        -   5. (iv) any combination of (i) to (iii); and, thereby,    -   b) treating cancer in a subject afflicted with cancer.

The method of treatment of the present invention, preferably, maycomprise steps in addition to those explicitly mentioned above. Forexample, further steps may relate, e.g., to localizing a tumor and/ordiagnosing cancer for step a), or administration of additionalmedication for step b). More preferably, the method of treatment furthercomprises the steps of the method for activating immune cells asspecified elsewhere herein, and the further step of administering saidactivated immune cells with antitumor activity to said subject.Moreover, one or more of said steps may be performed by automatedequipment. The method of the present invention, preferably, is an invivo method of treatment. Preferably, in the method of treatment, thecancer is a solid cancer, a metastasis, or a relapse thereof.

The term “treating” refers to ameliorating the diseases or disordersreferred to herein or the symptoms accompanied therewith to a,preferably, significant extent. Said treating as used herein alsoincludes an entire restoration of the health with respect to thediseases or disorders referred to herein. It is to be understood thattreating as used in accordance with the present invention may not beeffective in all subjects to be treated. However, the term shall,preferably, require that a statistically significant portion of subjectssuffering from a disease or disorder referred to herein can besuccessfully treated. Whether a portion is statistically significant canbe determined without further ado by the person skilled in the art usingvarious well known statistic evaluation tools, e.g., determination ofconfidence intervals, p-value determination, Student's t-test,Mann-Whitney test etc. Preferred confidence intervals are at least 90%,at least 95%, at least 97%, at least 98% or at least 99%. The p-valuesare, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, thetreatment shall be effective for at least 10%, at least 20% at least 50%at least 60%, at least 70%, at least 80%, or at least 90% of thesubjects of a given cohort or population. Preferably, treating cancer isreducing tumor burden in a subject. As will be understood by the skilledperson, effectiveness of treatment of e.g. cancer is dependent on avariety of factors including, e.g. cancer stage and cancer type.Preferably, treating cancer is reducing tumor burden.

Moreover, the present invention relates to a preparation of activatedimmune cells with antitumor activity obtained or obtainable by themethod for activating immune cells of the present invention.

Furthermore, the present invention relates to the use of

-   -   1. (i) a recombinant virus of the family Paramyxoviridae        according to the present invention,        -   2. (ii) a polynucleotide encoding the recombinant virus of            the family Paramyxoviridae according to (i), preferably a            polynucleotide according to an embodiment as specified            herein below;        -   3. (iii) a host cell comprising the recombinant virus of the            family Paramyxoviridae according to (i) and/or a            polynucleotide encoding the recombinant virus of the family            Paramyxoviridae according to (ii); preferably a host cell            according to an embodiment as specified herein below; or        -   4. (iv) any combination of (i) to (iii);    -   5. for the manufacture of a medicament, preferably for the        manufacture of a medicament for treating inappropriate cell        proliferation.

Also, the present invention relates to a recombinant virus of the familyParamyxoviridae according to the present invention and/or apolynucleotide according to the present invention and/or a host cellaccording to the present invention for use in medical treatment. Thepresent invention further relates to a recombinant virus of the familyParamyxoviridae according to the present invention and/or apolynucleotide according to the present invention and/or a host cellaccording to the present invention for use in treatment of inappropriatecell proliferation. Preferably, treatment of inappropriate cellproliferation is cancer treatment.

The present invention also relates to a kit comprising

-   -   1. (i) a recombinant virus of the family Paramyxoviridae        according of any one of claims 1 to 20,        -   2. (ii) a polynucleotide encoding the recombinant virus of            the family Paramyxoviridae according to (i), preferably a            polynucleotide according to an embodiment as specified            below;        -   3. (iii) a host cell comprising the recombinant virus of the            family Paramyxoviridae according to (i) and/or a            polynucleotide encoding the recombinant virus of the family            Paramyxoviridae according to (ii); preferably a host cell            according to an embodiment as specified below; or        -   4. (iv) any combination of (i) to (iii);        -   housed in a container.

The term “kit”, as used herein, refers to a collection of theaforementioned components. Preferably, said components are combined withadditional components, preferably within an outer container. The outercontainer, also preferably, comprises instructions for carrying out amethod of the present invention. Examples for components of the kit aswell as methods for their use have been given in this specification. Thekit, preferably, contains the aforementioned components in aready-to-use formulation. Preferably, the kit may additionally compriseinstructions, e.g., a user's manual for applying the recombinant virusof the family Paramyxoviridae with respect to the applications providedby the methods of the present invention. Details are to be foundelsewhere in this specification. Additionally, such user's manual mayprovide instructions about correctly using the components of the kit. Auser's manual may be provided in paper or electronic form, e.g., storedon CD or CD ROM. The present invention also relates to the use of saidkit in any of the methods according to the present invention.

Also, the present invention relates to a method for selecting (i) arecombinant virus of the family Paramyxoviridae comprising anexpressible polynucleotide encoding a tumor antigen or a fragment orvariant thereof, (ii) a polynucleotide encoding the recombinant virus of(i), and/or (iii) a host cell comprising the recombinant virus accordingto (i) and/or a polynucleotide according to (ii) for treating a subjectsuffering from cancer, said method comprising

-   -   a) detecting at least one tumor antigen expressed by cancer        cells in a cancer sample of said subject; and,    -   b) based on the determination of step a), selecting a        recombinant virus, a polynucleotide and/or a host cell        comprising an expressible polynucleotide encoding a tumor        antigen or a fragment or a variant thereof.

The method for selecting of the present invention, preferably, maycomprise steps in addition to those explicitly mentioned above. Forexample, further steps may relate, e.g., a cancer sample for step a), oradministration of a recombinant virus, a polynucleotide and/or a hostcell comprising an expressible polynucleotide encoding a tumor antigenor a fragment or a variant thereof to a cancer sample after step b).Moreover, one or more of said steps may be performed by automatedequipment. The method of the present invention, preferably, is an invitro method, preferably an in vitro method aiding in taking a treatmentdecision.

As used herein, the term “detecting a tumor antigen” relates todetecting the presence of a tumor antigen in and/or on the surface of acell, preferably cancer cell. Preferably, detection is qualitative, morepreferably semi-quantitative, most preferably quantitative detection.Preferably, detecting is immunologically detecting, preferably bycontacting a, preferably isolated, cancer sample or a fraction thereofwith at least one antibody binding to the tumor antigen; preferably,said binding is specific binding. As used herein, the term “specificbinding” relates to a binding in which other compounds which are not thetumor antigen are bound with an affinity at least 100 fold, morepreferably at least 1000 fold, most preferably at least 10⁴ fold loweras compared to the affinity for the tumor antigen. Preferred methods ofimmunologically detecting a tumor antigen are known in the art andinclude in particular in-situ immunostaining, ELISA and related methods,and immunoblots. As will be understood by the skilled person, expressionof a tumor antigen may also be detected by detecting an RNA encodingsaid tumor antigen expressed by a tumor cell.

Selection of the agent for treating a subject suffering from cancer,i.e. the specific selection of recombinant virus, polynucleotide and/orhost cell comprising an expressible polynucleotide encoding a tumorantigen or a fragment or a variant thereof, will depend on the specificcircumstances. Preferably, if it is found that cancer cells in a cancersample express a tumor antigen, an agent increasing expression of saidtumor antigen will be selected. On the other hand, in case it is foundthat the cancer cells express a mutant form of a polypeptide (e.g. aneoantigen), preferably, an agent inducing expression of said mutantform will be elected. Thus, preferably the tumor antigen or fragment orvariant thereof encoded by the expressible polynucleotide comprised inthe recombinant virus, the polynucleotide and/or the host cell selectedin step b) shares at least one epitope with the tumor antigen detectedin step a). Also preferably, the tumor antigen or fragment or variantthereof encoded by the expressible polynucleotide comprised in therecombinant virus, the polynucleotide and/or the host cell selected instep b) is or is derived from the tumor antigen detected in step a).

In view of the above, the following embodiments are particularlyproposed:

EMBODIMENTS

1. A recombinant virus of the family Paramyxoviridae comprising anexpressible polynucleotide encoding at least one of (i) a tumor antigen,(ii) a fragment of a tumor antigen, and (iii) a variant of (i) or (ii).

2. The recombinant virus of the family Paramyxoviridae of embodiment 1,wherein said tumor antigen is selected from the group consisting ofL-dopachrome-tautomerase (TRP2), melanocyte protein PMEL (gp100), HPVE6/7, MAGE 1, MAGE 3, NY-ESO, androgen receptor (AR), BCL-1,calprotectin, carcinoembryonic antigen (CEA), EGFRs, epithelial celladhesion molecule (Ep-CAM), epithelial sialomucin, membrane estrogenreceptors (mER), FAP HER2/neu, human high molecular weightmelanoma-associated antigen (HMW-MAA), IL-6, MOC-1, MOC-21, MOC-52,melan-A/MART-1, melanoma-associated antigen, mucin, OKT9, progesteronereceptor (PGR), prostate specific antigen (PSA), prostate stem cellantigen (PSCA), prostate-specific membrane antigen (PSMA),symaptophysin, VEGFRs, CD19, CD20, CD22, CD30 and CD33. More preferably,the tumor antigen is HPV E6, HPVE7, MAGE 1, MAGE 3, NY-ESO, TRP2 orgp100, more preferably is TRP2.

3. The recombinant virus of the family Paramyxoviridae of embodiment 1or 2, wherein said tumor antigen is a human tumor antigen.

4. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 3, wherein said fragment comprises at least five,preferably at least six, more preferably at least seven, even morepreferably at least eight, most preferably at least nine amino acids,wherein preferably, said amino acids correspond to a contiguous sequencecomprised in the amino acid sequence of said tumor antigen.

5. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 4, wherein said fragment comprises at least oneantigenic epitope of said tumor antigen.

6. The recombinant virus of the family Paramyxoviridae of embodiment 5,wherein said epitope is an MHC class I epitope and/or an MHC class IIepitope.

7. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 6, wherein said fragment is selected from the groupconsisting of (i) a fragment of a human papillomavirus (HPV) E6polypeptide, preferably of an E6 polypeptide of a high-risk HPV, e.g. ofa HPV16 E6 (Genbank Acc No: NP_041325.1 GI:9627104), (ii) a fragment ofa HPV E7 polypeptide, preferably of an E7 polypeptide of a high-riskHPV, e.g. of a HPV16 E7 (Genbank Acc No: NP_041326.1 GI:9627105); (iii)TRP2, preferably human TRP2 (preferably. encoded by Genbank Acc No: NM001922.4 GI:1015809739), (iv) cancer/testis antigen 1B (CTAG1B, alsoreferred to as NY-ESO, preferably encoded by Genbank Acc No: NM 001327.2GI:215272337, an (v) an arbitrary combination of any of (i) to (iv).

8. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 7, wherein said tumor antigen, fragment or variantthereof comprises, preferably consists of, at least one of (i) a HPV E6polypeptide, preferably of an E6 polypeptide of a high-risk HPV, e.g. ofa HPV16 E6 (Genbank Acc No: NP_041325.1 GI:9627104), (ii) a HPV E7polypeptide, preferably of an E7 polypeptide of a high-risk HPV, e.g. ofa HPV16 E7 (Genbank Acc No: NP_041326.1 GI:9627105); (iii) TRP2,preferably human TRP2 (preferably. encoded by Genbank Acc No: NM001922.4 GI:1015809739), (iv) cancer/testis antigen 1B (CTAG1B, alsoreferred to as NY-ESO, preferably encoded by Genbank Acc No: NM 001327.2GI:215272337), and (v) an arbitrary combination of any of (i) to (iv).

9. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 8, wherein said expressible polynucleotide encodes atleast one further of (i) a tumor antigen, (ii) a fragment of a tumorantigen, and (iii) a variant of (i) or (ii).

10. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 9, wherein said expressible polynucleotide is comprisedin the genome of said recombinant virus, preferably in a regioncorresponding to the region intervening the H and the L gene of measlesvirus.

11. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 10, further comprising at least one expressiblepolynucleotide encoding a further activator of the immune response.

12. The recombinant virus of the family Paramyxoviridae of embodiment11, wherein said further activator of the immune response comprises,preferably is, an immunoglobulin or fragment thereof.

13. The recombinant virus of the family Paramyxoviridae of embodiments11 or 12, wherein said further activator of the immune responsecomprises, preferably is, a secreted soluble activator of the immuneresponse, preferably IL-12.

14. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 11 to 13, wherein said further activator of the immuneresponse comprises, preferably is, a secreted immunoglobulin or fragmentthereof.

15. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 11 to 14, wherein said further activator of the immuneresponse comprises, preferably is, a single-chain antibody or ananobody.

16. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 11 to 15, wherein said further activator of the immuneresponse comprises, preferably is, a secreted soluble anti-PD-1/PD-L1antibody.

17. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 16, wherein said recombinant virus is a recombinantMorbillivirus, preferably, a recombinant measles virus (MV).

18. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 17, wherein said recombinant MV is derived from MVstrain Edmonston A or B, preferably B, more preferably from MV vaccinestrain Schwarz/Moraten.

19. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 18, wherein said at least one expressiblepolynucleotide is comprised in a polynucleotide encoding the recombinantvirus of the family Paramyxoviridae.

20. The recombinant virus of the family Paramyxoviridae of any one ofembodiments 1 to 19, wherein said polynucleotide encoding therecombinant virus of the family Paramyxoviridae comprises, preferablyconsists of, the nucleic acid sequence of any one of SEQ ID NOs: 3 to 5.

21. A polynucleotide encoding the recombinant virus of the familyParamyxoviridae according to any one of embodiments 1 to 20.

22. The polynucleotide according to embodiment 21, wherein saidpolynucleotide comprises, preferably consists of, the nucleic acidsequence any one of SEQ ID NOs: 3 to 5.

23. A host cell comprising the recombinant virus of the familyParamyxoviridae according to any one of embodiments 1 to 20 and/or thepolynucleotide encoding the recombinant virus of the familyParamyxoviridae according to embodiment 21 or 22.

24. The host cell of embodiment 23, wherein said host cell is a cancercell.

25. A medicament comprising

(a) (i) a recombinant virus of the family Paramyxoviridae according ofany one of embodiments 1 to 20,

-   -   (ii) a polynucleotide encoding the recombinant virus of the        family Paramyxoviridae according to (i), preferably a        polynucleotide according to embodiment 21 or 22;    -   (iii) a host cell comprising the recombinant virus of the family        Paramyxoviridae according to (i) and/or a polynucleotide        encoding the recombinant virus of the family Paramyxoviridae        according to (ii); preferably a host cell according to        embodiment 23 or 24; or    -   (iv) any combination of (i) to (iii); and

(b) at least one pharmacologically acceptable excipient.

26. A method for activating immune cells with antitumor activity in asample comprising cancer cells and immune cells, comprising

-   -   a) contacting said sample comprising cancer cells and immune        cells with

(i) a recombinant virus of the family Paramyxoviridae according to anyone of embodiments 1 to 20,

(ii) a polynucleotide encoding the recombinant virus of the familyParamyxoviridae according to (i), preferably a polynucleotide accordingto embodiment 21 or 22;

(iii) a host cell comprising the recombinant virus of the familyParamyxoviridae according to (i) and/or a polynucleotide encoding therecombinant virus of the family Paramyxoviridae according to (ii);preferably a host cell according to embodiment 23 or 24; or

(iv) any combination of (i) to (iii); and thereby,

-   -   b) activating immune cells with antitumor activity comprised in        said sample.

27. The method of embodiment 26, wherein said method is an in vitromethod. 28. A method for treating cancer in a subject afflicted withcancer, comprising

-   -   a) contacting said subject with

(i) a recombinant virus of the family Paramyxoviridae according to anyone of embodiments 1 to 20,

(ii) a polynucleotide encoding the recombinant virus of the familyParamyxoviridae according to (i), preferably a polynucleotide accordingto embodiment 21 or 22;

(iii) a host cell comprising the recombinant virus of the familyParamyxoviridae according to (i) and/or a polynucleotide encoding therecombinant virus of the family Paramyxoviridae according to (ii);preferably a host cell according to embodiment 23 or 24; or

(iv) any combination of (i) to (iii); and, thereby,

-   -   b) treating cancer in a subject afflicted with cancer.

29. The method of embodiment 28, wherein said cancer is a solid cancer,a metastasis, or a relapse thereof.

30. The method of embodiment 28 or 29, wherein treating cancer isreducing tumor burden.

31. The method of any one of embodiments 28 to 30, wherein said canceris malignant melanoma, head and neck cancer, hepatocellular carcinoma,pancreatic carcinoma, prostate cancer, renal cell carcinoma, gastriccarcinoma, colorectal carcinoma, a lymphoma or a leukemia.

32. The method of any one of embodiments 28 to 31, wherein said methodfurther comprises the steps of the method for activating immune cellswith antitumor activity in a sample according to embodiment 26, and thefurther step of administering said activated immune cells with antitumoractivity to said subject.

33. A preparation of activated immune cells with antitumor activityobtained or obtainable by the method of embodiment 26 or 27.

34. Use of

(i) a recombinant virus of the family Paramyxoviridae according to anyone of embodiments 1 to 20,

(ii) a polynucleotide encoding the recombinant virus of the familyParamyxoviridae according to (i), preferably a polynucleotide accordingto embodiment 21 or 22;

(iii) a host cell comprising the recombinant virus of the familyParamyxoviridae according to (i) and/or a polynucleotide encoding therecombinant virus of the family Paramyxoviridae according to (ii);preferably a host cell according to embodiment 23 or 24; or

(iv) any combination of (i) to (iii);

-   -   for the manufacture of a medicament, preferably for the        manufacture of a medicament for treating inappropriate cell        proliferation.

35. A recombinant virus of the family Paramyxoviridae according to anyone of embodiments 1 to 20 and/or a polynucleotide according toembodiment 21 or 22 and/or a host cell according to embodiment 23 or 24for use in medical treatment.

36. A recombinant virus of the family Paramyxoviridae according to anyone of embodiments 1 to 20 and/or a polynucleotide according toembodiment 21 or 22 and/or a host cell according to embodiment 23 or 24for use in treatment of inappropriate cell proliferation.

37. The recombinant virus of the family Paramyxoviridae for use ofembodiment 36, wherein treatment of inappropriate cell proliferation iscancer treatment.

38. A kit comprising

(i) a recombinant virus of the family Paramyxoviridae according of anyone of embodiments 1 to 20,

(ii) a polynucleotide encoding the recombinant virus of the familyParamyxoviridae according to (i), preferably a polynucleotide accordingto embodiment 21 or 22;

(iii) a host cell comprising the recombinant virus of the familyParamyxoviridae according to (i) and/or a polynucleotide encoding therecombinant virus of the family Paramyxoviridae according to (ii);preferably a host cell according to embodiment 23 or 24; or

(iv) any combination of (i) to (iii);

-   -   housed in a container.

39. A method for selecting (i) a recombinant virus of the familyParamyxoviridae comprising an expressible polynucleotide encoding atumor antigen or a fragment or variant thereof, (ii) a polynucleotideencoding the recombinant virus of (i), and/or (iii) a host cellcomprising the recombinant virus according to (i) and/or apolynucleotide according to (ii) for treating a subject suffering fromcancer, said method comprising

a) detecting at least one tumor antigen expressed by cancer cells in acancer sample of said subject; and,

b) based on the determination of step a), selecting a recombinant virus,a polynucleotide and/or a host cell comprising an expressiblepolynucleotide encoding a tumor antigen or a fragment or a variantthereof.

40. The method of embodiment 39, wherein the tumor antigen or fragmentor variant thereof encoded by the expressible polynucleotide comprisedin the recombinant virus, the polynucleotide and/or the host cellselected in step b) shares at least one epitope with the tumor antigendetected in step a).

41. The method of embodiment 39 or 40, wherein the tumor antigen orfragment or variant thereof encoded by the expressible polynucleotidecomprised in the recombinant virus, the polynucleotide and/or the hostcell selected in step b) is or is derived from the tumor antigendetected in step a).

42. The method of any one of embodiments 39 to 41, wherein therecombinant virus of (i) is a recombinant virus according to any one ofembodiments 1 to 20,

wherein the polynucleotide of (ii) is a polynucleotide according toembodiment 21 or 22, and/or wherein the host cell of (iii) is a hostcell according to embodiment 23 or 24.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C: FIG. 1A) Schematic representation of the recombinant MVgenome. cDNA encoding trp2 (1667 bp) was inserted into the MeV Schwarzgenome into an additional transcription unit downstream of thehemagglutinin open reading frame. FIG. 1B) Upper panel: growth ofmeasles virus constructs (MV-trp2 or MV (control)) on Vero cells;x-axis: time (hours), y-axis: titer (cell infectious units (ciu)/ml);lower panel: viability of host cells over the course of MV-trp2 and MV(control) replication, as compared to a mock-infected culture; x-axis:Time (hours), y-axis: relative viability compared to mock infectedculture. FIG. 1C) Expression of trp2 by MV-trp2, but not by MV; upperpanel: RT-PCR with trp2-specific primers; lower panel: immunoblot withanti-trp2 antibodies.

FIG. 2 : IFN-gamma ELISPOT analysis of co-cultures described in Example2; upper panels: photographs of culture dishes after ELISPOT assay;lower graph: quantification of the spots in culture dishes as shown inthe upper panels.

FIG. 3 : Eliciting anti-tumor immunity in mice by intratumoral injectionof MV-trp2 (Example 3); upper panel: Treatment schedule; lower leftpanel: quantification of results of an IFN-gamma ELISPOT analysis usingsplenocytes of mice from different treatment groups in co-culture withMC38-hCD46 cells infected with MV-trp2 or parental MV; lower rightpanel: quantification of results of an IFN-gamma ELISPOT analysis usingsplenocytes of mice from different treatment groups in co-culture withB16 cells.

The following Examples shall merely illustrate the invention. They shallnot be construed, whatsoever, to limit the scope of the invention.

Example 1

Recombinant measles viruses encoding the tumor-associated antigen (TAA)trp2 have been generated according to known methods (MV-trp2, FIG. 1A).

Vero cells were transduced with parental MV or MV encoding trp2 with amultiplicity of infection (MOI)=3. At designated time points, cells wereharvested and progeny viral particles were determined in titrationassays by serial dilution. Insertion of the tumor-associated antigendoes not impair viral replication and cytotoxic effects (FIG. 1B, upperpanel). At designated time points, cell viability was determined using acolorimetric XTT assay (FIG. 1B, lower panel). Mock treated cells wereused as a reference for viability=1.0.

RNA was extracted from Vero cells infected with parental MV or MVencoding trp2. cDNA synthesis was performed using oligo-dT primers. PCRwas performed with trp2-specific primers. Cells infected with parentalMV or MV encoding trp2 were lysed in RIPA buffer for protein extraction.After SDS PAGE of cell lysates, Western blot analysis was performed withtrp2-specific antisera. Beta-actin was detected as a loading control. Amelanosome preparation was used as a positive control. Tumor-antigenexpression in infected cells was confirmed both on the mRNA- and proteinlevel (FIG. 1C).

Example 2

Trp2-specific T cells were co-cultured with MC38-hCD46 cells infectedwith MV-trp2 or parental MV or with B16 cells or stimulated with theimmunodominant trp2 peptide. After 16 hours of co-culture, IFN-gammaELISPOT analysis was performed. MV encoding the trp2 tumor antigen (TAA)activate TAA-specific T cells: Co-culture of trp2-specific T cells withmurine cells infected with MV-trp2 leads to significantly higherIFN-gamma secretion than co-culture with parental MV (FIG. 2 ).

Example 3

1×10⁶ B16ova hCD46 cells were implanted into the flank of C57BL/6J mice(day 0). Starting on day 10, mice received intratumoral injections of1×10⁶ cell infectious units (ciu) of MV-trp2, 1×10⁶ ciu of parental MVin a total volume of 1004 or 1004 carrier fluid (mock). Mice weresacrificed on day 23 and spleens were extracted for ELISPOT analysis.

Splenocytes of mice from different treatment groups were co-culturedwith MC38-hCD46 cells infected with MV-trp2 or parental MV. After 16hours of co-culture, IFN-gamma ELISPOT analysis was performed.Splenocytes of mice from different treatment groups were co-culturedwith B16 cells. After 16 hours of co-culture, IFN-gamma ELISPOT analysiswas performed. It was found that intratumoral injection of MV-trp2 canenhance TAA-specific and tumor-specific immunity (FIG. 3 ).

Thus, in the present invention, an oncolytic MV vaccine was generated toelicit a tumour antigen-specific immune response. With this approach,the MV vector encoded tumour antigen is expressed in the context ofviral danger-associated molecular patterns (DAMPs) andpathogen-associated molecular patterns (PAMPs) which serve as “naturaladjuvants”. Furthermore, MV-mediated oncolysis functions as an in situtumour vaccine: MV infection causes immunogenic cell death within thetumour accompanied by release of tumour-associated antigens, resultingin efficient antigen cross-presentation and epitope spread

1-42. (canceled)
 43. A replication competent recombinant virus of thefamily Paramyxoviridae comprising an expressible polynucleotide encodingat least one of (a) the tumor antigen L-dopachrome-tautomerase (TRP2),(b) a fragment of the tumor antigen TRP2 comprising at least oneantigenic epitope of TRP2, and (c) a variant of (a) or (b), wherein thevariant is 90% identical to the sequence of TRP2 depicted in SEQ ID NO:2.
 44. The recombinant virus of the family Paramyxoviridae of claim 43,wherein said fragment of the tumor antigen TRP2 comprising at least oneantigenic epitope of TRP2 comprises a sequence of from 7 to 15contiguous amino acids.
 45. The recombinant virus of the familyParamyxoviridae of claim 43, wherein TRP2 is human TRP2.
 46. Therecombinant virus of the family Paramyxoviridae of claim 43, whereinsaid recombinant virus is a recombinant Morbillivirus.
 47. Therecombinant virus of the family Paramyxoviridae of claim 46, whereinsaid recombinant Morbillivirus is a recombinant measles virus (MV). 48.The recombinant virus of the family Paramyxoviridae of claim 43, furthercomprising at least one expressible polynucleotide encoding a furtheractivator of the immune response.
 49. The recombinant virus of thefamily Paramyxoviridae of claim 48, wherein said at least oneexpressible polynucleotide encoding a further activator of the immuneresponse is comprised in a polynucleotide encoding the recombinant virusof the family Paramyxoviridae.
 50. A polynucleotide encoding thereplication competent recombinant virus of claim
 43. 51. A host cellcomprising the replication competent recombinant virus of claim
 43. 52.A method for activating immune cells with antitumor activity in a samplecomprising cancer cells and immune cells, comprising: a) contacting saidsample comprising cancer cells and immune cells with (i) the replicationcompetent recombinant virus of the family Paramyxoviridae of claim 43;(ii) a polynucleotide encoding the recombinant virus of the familyParamyxoviridae of a)(i); (iii) a host cell comprising the recombinantvirus of the family Paramyxoviridae of a)(i); (iv) a host cellcomprising the polynucleotide of a)(ii); or (v) any combination of a)(i)to a)(iv); and thereby, b) activating immune cells with antitumoractivity comprised in said sample.
 53. A preparation of activated immunecells with antitumor activity obtained or obtainable by the method ofclaim
 52. 54. A method for treating inappropriate cell proliferationcomprising administering to a subject in need thereof: (i) thereplication competent recombinant virus of the family Paramyxoviridae ofclaim 43; (ii) a polynucleotide encoding the replication competentrecombinant virus of the family Paramyxoviridae of (i); (iii) a hostcell comprising the replication competent recombinant virus of thefamily Paramyxoviridae of (i); (iv) a host cell comprising thepolynucleotide of (ii); or (v) any combination of (i) to (iv).
 55. Themethod of claim 54, wherein said inappropriate cell proliferation iscancer.
 56. A kit comprising (i) the replication competent recombinantvirus of the family Paramyxoviridae of claim 43; (ii) a polynucleotideencoding the replication competent recombinant virus of the familyParamyxoviridae of (i); (iii) a host cell comprising the replicationcompetent recombinant virus of the family Paramyxoviridae of (i); (iv) ahost cell comprising the polynucleotide of (ii); or (v) any combinationof (i) to (iv); housed in a container.
 57. A host cell comprising thepolynucleotide according to claim 51.